1a.Objectives (from AD-416):
To screen a previously constructed Bacterial Artificial Chromosome (BAC) library, derived from western corn rootworm (WCR) and held in the Cooperator's laboratory, with 50 validated Single Nucleotide Polymorphism (SNP) loci to determine which clones carry each locus. To prepare DNA from selected BAC clones for high throughput sequencing. To use DNA from backcross pedigrees of WCR to create a genetic linkage map for organophosphate insecticide resistance.

1b.Approach (from AD-416):
A subset of 50 validated Single Nucleotide Polymorphism (SNP) loci derived from Expressed Sequence Tag (EST) sequence data will be used to screen the available Western Corn Rootworm (WCR) Bacterial Artificial Chromosome (BAC) library prepared previously by the Cooperator to determine which clones carry each locus. Screening will be done by PCR by USDA researchers in Ames, IA. Each superpool contains DNA from 4,608 clones corresponding to twelve 384-well plates of library clones, giving a total of 24 superpools for the entire library. For each superpool, there is an associated "pool plate" that contains 12 pools of DNA from each 384-well plate, 16 pools from each row of all the plates, 24 pools from each column of all the plates, and 24 "diagonal pools." Thus, a PCR amplicon can be associated with an individual BAC using 100 PCR assays (24 to identify the superpool plus 76 to determine the plate, row, and column within the superpool). A genetic linkage map will be constructed with approximately 600 SNP markers developed by USDA ARS, using backcross pedigrees of WCR that were collected by the Cooperator and phenotyped for organophosphate resistance. The set of SNP loci to be used in screening will be selected to give an even distribution across the linkage map, but may also target loci for which EST annotations or other information indicates a gene relevant to adaptations to pest management or potential biotechnology applications. PCR primers will be designed from the EST sequences from which the SNP was derived. Optimal PCR conditions for each primer pair will be determined using WCR genomic DNA as a template. In cases where optimal conditions cannot be determined, new primer pairs will be designed and tested. For each SNP locus an initial round of 24 PCR assays followed by agarose gel electrophoresis of the reaction products will determine the superpools that contain the amplicon. Second-round PCR assays and agarose gel electrophoresis on the relevant pool plates will identify the BAC clones containing the amplicon. Because the BAC library is estimated to give 4.6-fold coverage of the WCR genome, we expect, on average, to perform five second-round assays per SNP locus. BAC clones that have been associated with a SNP locus will be digested with HindIII, BamHI, or EcoRI restriction enzymes and separated by agarose gel electrophoresis. The resulting restriction-site fingerprints will be used to arrange the BAC clones associated with each SNP locus into a contiguous region. DNA will be extracted from 1 or 2 BAC clones from each contig associated with a SNP locus for future high-throughput 454 (Roche) pyrosequencing. The clones to be sequenced will be selected to maximize the length of sequence obtained on each side of the SNP.

3.Progress Report:

A Western Corn Rootworm (WCR) Bacterial Artificial Chromosome (BAC) library was constructed. A BAC is a type of tool, called a vector, that inserts long stretches of DNA from any species into a bacterium. Once the foreign DNA has been cloned into the bacterium, many copies of it can be made and sequenced. The library we constructed contains 110,592 clones of long stretches of DNA from the WCR. DNA preparations representing the entire library, organized into pools and "superpools," were screened for genes of interest using a technique that greatly amplifies a single to a few pieces of DNA. Each superpool contains DNA from 4,608 clones corresponding to 12 384-well plates of library clones, giving a total of 24 superpools for the entire library. For each superpool, there is an associated pool plate containing 12 pools of DNA from each 384-well plate, 16 pools from each row of all the plates, 24 pools from each column of all the plates and 24 diagonal pools. This kind of arrangement allows efficient screening for particular genes of interest so that a bacterial artificial chromosome clone can be identified and sequenced in its entirety. This library was screened to identify gene coding for important proteins such as receptors and enzymes that are often involved in insecticide resistance. To date, 18 WCR BAC library clones were successfully identified as containing specific genes of interest. DNA from these clones was extracted and sequenced. In addition to providing direct data about the target genes, information from sequence analysis has been useful in characterizing the overall structure of the WCR genome which has made it possible to begin a project to sequence the whole genome. Recent screening has identified approximately ten additional clones putatively containing other genes of interest. A genetic linkage map has been constructed using the markers validated in the parent research project. The linkage map shows the relative position of genes, with the genes closer together being more likely to be inherited together. The library is now being screened further with the SNP markers, and 70 of these BACs will be strategically selected for full sequencing based on the new linkage map that the markers have made possible.